The vascular endothelium releases a variety of vasoactive substances, including the endothelium-derived vasoconstrictor peptide, endothelin (ET) (see, e.g., Vanhoutte et al. (1986) Annual Rev. Physiol. 48: 307-320; Furchgott and Zawadski (1980) Nature 288: 373-376). Endothelin, which was originally identified in the culture supernatant of porcine aortic endothelial cells (see, Yanagisawa et al. (1988) Nature 332:411-415), is a potent twenty-one amino acid peptide vasoconstrictor. It is the most potent vasopressor known and is produced by numerous cell types, including the cells of the endothelium, trachea, kidney and brain. Endothelin is synthesized as a two hundred and three amino acid precursor preproendothelin that contains a signal sequence which is cleaved by an endogenous protease to produce a thirty-eight (human) or thirty-nine (porcine) amino acid peptide. This intermediate, referred to as big endothelin, is processed in vivo to the mature biologically active form by a putative endothelin-converting enzyme (ECE) that appears to be a metal-dependent neutral protease (see, e.g., Kashiwabara et al. (1989) FEBS Lttrs. 247: 337-340). Cleavage is required for induction of physiological responses (see, e.g., von Geldern et al. (1991) Peptide Res. 4: 32-35). In porcine aortic endothelial cells, the thirty-nine amino acid intermediate, big endothelin, is hydrolyzed at the Trp.sup.21 -Val.sup.22 bond to generate endothelin-1 and a C-terminal fragment. A similar cleavage occurs in human cells from a thirty-eight amino acid intermediate. Three distinct endothelin isopeptides, endothelin-1, endothelin-2 and endothelin-3, that exhibit potent vasoconstrictor activity have been identified.
The family of three isopeptides endothelin-1, endothelin-2 and endothelin-3 are encoded by a family of three genes (see, Inoue et al. (1989) Proc. Natl. Acad. Sci. USA 86: 2863-2867; see, also Saida et al. (1989) J. Biol. Chem. 264: 14613-14616). The nucleotide sequences of the three human genes are highly conserved within the region encoding the mature 21 amino acid peptides and the C-terminal portions of the peptides are identical. Endothelin-2 is (Trp.sup.6, Leu.sup.7) endothelin-1 and endothelin-3 is (Thr.sup.2, Phe.sup.4, Thr.sup.5,Tyr.sup.6, Lys.sup.7, Tyr.sup.14) endothelin-1. These peptides are, thus, highly conserved at the C-terminal ends.
Release of endothelins from cultured endothelial cells is modulated by a variety of chemical and physical stimuli and appears to be regulated at the level of transcription and/or translation. Expression of the gene encoding endothelin-1 is increased by chemical stimuli, including adrenaline, thrombin and Ca.sup.2+ ionophore. The production and release of endothelin from the endothelium is stimulated by angiotensin II, vasopressin, endotoxin, cyclosporine and other factors (see, Brooks et al. (1991) Eur. J. Pharm. 194:115-117), and is inhibited by nitric oxide. Endothelial cells appear to secrete short-lived endothelium-derived relaxing factors (EDRF), including nitric oxide or a related substance (Palmer et al. (1987) Nature 327: 524-526), when stimulated by vasoactive agents, such as acetylcholine and bradykinin. Endothelin-induced vasoconstriction is also attenuated by atrial natriuretic peptide (ANP).
The endothelin peptides exhibit numerous biological activities in vitro and in vivo. Endothelin provokes a strong and sustained vasoconstriction in vivo in rats and in isolated vascular smooth muscle preparations; it also provokes the release of eicosanoids and endothelium-derived relaxing factor (EDRF) from perfused vascular beds. Intravenous administration of endothelin-1 and in vitro addition to vascular and other smooth muscle tissues produce long-lasting pressor effects and contraction, respectively (see, e.g., Bolger et al. (1991) Can. J. Physiol. Pharmacol. 69:406-413). In isolated vascular strips, for example, endothelin-1 is a potent (EC.sub.50 =4.times.10.sup.-10 M), slow acting, but persistent, contractile agent. In vivo, a single dose elevates blood pressure in about twenty to thirty minutes. Endothelin-induced vasoconstriction is not affected by antagonists to known neurotransmitters or hormonal factors, but is abolished by calcium channel antagonists, The effect of calcium channel antagonists, however, is most likely the result of inhibition of calcium influx, since calcium influx appears to be required for the long-lasting contractile response to endothelin.
Endothelin also mediates renin release, stimulates ANP release and induces a positive inotropic action in guinea pig atria. In the lung, endothelin-1 acts as a potent bronchoconstrictor (Maggi et al. (1989) Eur. J. Pharmacol. 160: 179-182). Endothelin increases renal vascular resistance, decreases renal blood flow, and decreases glomerular filtrate rate. It is a potent mitogen for glomerular mesangial cells and invokes the phosphoinoside cascade in such cells (Simonson et al. (1990) J. Clin. Invest. 85: 790-797).
There are specific high affinity binding sites (dissociation constants in the range of 2-6.times.10.sup.-10 M) for the endothelins in the vascular system and in other tissues, including the intestine, heart, lungs, kidneys, spleen, adrenal glands and brain. Binding is not inhibited by catecholamines, vasoactive peptides, neurotoxins or calcium channel antagonists. Endothelin binds and interacts with receptor sites that are distinct from other autonomic receptors and voltage dependent calcium channels. Competitive binding studies indicate that there are multiple classes of receptors with different affinities for the endothelin isopeptides. The sarafotoxins, a group of peptide toxins from the venom of the snake Atractaspis einqadensis that cause severe coronary vasospasm in snake bite victims, have structural and functional homology to endothelin-1 and bind competitively to the same cardiac membrane receptors (Kloog et al. (1989) Trends Pharmacol. Sci. 10:212-214).
Two distinct endothelin receptors, designated ET.sub.A and ET.sub.B, have been identified and there is evidence that other subtypes exist (see, e.g., Emori et al. (1990) FEBS Lett. 263:261-264; and Sokolovsky et al. (1992) J. Biol. Chem. 267:20551-20554). DNA clones encoding the ET.sub.A and ET.sub.B receptors have been isolated (Arai et al. (1990) Nature 348: 730-732; Sakurai et al. (1990) Nature 348: 732-735). Based on the amino acid sequences of the proteins encoded by the cloned DNA, it appears that each receptor contains seven membrane spanning domains and exhibits structural similarity to G-protein-coupled membrane proteins. Messenger RNA encoding both receptors has been detected in a variety of tissues, including heart, lung, kidney and brain.
The distribution of receptor subtypes is tissue specific (Martin et al. (1989) Biochem. Biophys. Res. Commun. 162: 130-137) and the affinity of each receptor for members of the endothelin family of peptides can be distinguished. ET.sub.A receptors appear to be selective for endothelin-1 and are predominant in cardiovascular tissues. ET.sub.B receptors are predominant in noncardiovascular tissues, including the central nervous system and kidney, and interact with the three endothelin isopeptides (Sakurai et al. (1990) Nature 348: 732-734). In addition, ET.sub.A receptors occur on vascular smooth muscle, are linked to vasoconstriction and have been associated with cardiovascular, renal and central nervous system diseases; whereas ET.sub.B receptors are located on the vascular endothelium, linked to vasodilation (Takayanagi et al. (1991) FEBS Lett. 282: 103-106) and have been associated with bronchoconstrictive disorders. The ET.sub.A receptor appears to mediate the principal part of the vasoconstriction induced by ET-1 (Ihara et al. (1993) Lif. Sci 50:247-255) and the ET.sub.B subtype mediates endothelium-dependent vasodilation (Takayanagi et al. (1991) FEBS Lett. 282:103-106), Selective agonist-induced stimulation of ET.sub.B, however, can induce vasoconstriction (see, e.g., MCMurdo et al. (1993) Br. J. Pharmac. 108:557-561; and Moreland et al. (1992) Biochem. Biophys. Res. Commun. 184:100-106).
By virtue of the distribution of receptor types and the differential affinity of each isopeptide for each receptor type, the activity of the endothelin isopeptides varies in different tissues. For example, endothelin-1 inhibits .sup.125 I-labelled endothelin-1 binding in cardiovascular tissues forty to seven hundred times more potently than endothelin-3. .sup.125 I-labelled endothelin-1 binding in non-cardiovascular tissues, such as kidney, adrenal gland, and cerebellum, is inhibited to the same extent by endothelin-1 and endothelin-3, which indicates that ET.sub.A receptors predominate in cardiovascular tissues and ET.sub.B receptors predominate in non-cardiovascular tissues.
Endothelin plasma levels are elevated in certain disease states. Endothelin-1 plasma levels in healthy individuals, as measured by radioimmunoassay (RIA), are about 0.26-5 pg/ml. Blood levels of endothelin-1 and its precursor, big endothelin, are elevated in shock, myocardial infarction, vasospastic angina, kidney failure and a variety of connective tissue disorders. In patients undergoing hemodialysis or kidney transplantation or suffering from cardiogenic shock, myocardial infarction or pulmonary hypertension levels are as high as 35 pg/ml have been observed (see, Stewart et al. (1991 ) Annals Internal Med. 114: 464-469). Because endothelin is likely to be a local, rather than a systemic, regulating factor, it is probable that the levels of endothelin at the endothelium/smooth muscle interface are much higher than circulating levels.
Endothelin agonists and antagonists
Because endothelin is associated with certain disease states and is implicated in numerous physiological effects, compounds that can interfere with or potentiate endothelin-associated activities, such as endothelin-receptor interaction and vasoconstrictor activity, are of interest. A number of compounds that exhibit endothelin antagonistic activity have been identified. These include cyclic pentapeptides, acyltripeptides, hexapeptide analogs, certain antraquinone derivatives, indanecarboxylic acids, certain N-pyriminylbenzenesulfonamides, certain benzenesulfonamides, and certain naphthalenesulfonamides (Nakajima et al. (1991) J. Antibiot. 44:1348-1356; Miyata et al. (1992) J, Antibiot. 45:74-8; Ishikawa et al. (1992) J. Med. Chem. 35:2139-2142; U.S. Pat. No. 5,114,918 to Ishikawa et al.; EP A10 569 193; EP A10 558 258; EP A10 436 189 to BANYU PHARMACEUTICAL CO., LTD (Oct. 7, 1991); Canadian Patent Application 2,067,288; Canadian Patent Application 2,071,193; U.S. Pat. No. 5,208,243; U.S. Pat. No. 5,270,313; Cody et al. (1993) Med. Chem. Res. 3:154-162; Miyata et al. (1992) J. Antiblot 45:1041-1046; Miyata et al. (1992) J. Antibiot 45:1029-1040, Fujimoto et al. (1992) FEBS Lett. 305:41-44; Oshashi et al. (1002) J. Antiblot 45:1684-1685; EP A10 496 452; Clozel et al. (1993) Nature 365:759-761; International Patent Application WO93/08799; Nishikibe et al. (1993) Life Sci. 52:717-724; and Benigni et al. (1993) Kidney Int. 44:440-444).
In particular, a fermentation product of Streptomyces misakiensis, designated BE-18257B, has been identified as an ET.sub.A receptor antagonist. BE-18257B is a cyclic pentapeptide, cyclo(D-Glu-L-Ala-allo-D-Ile-L-Leu-D-Trp), which inhibits .sup.125 I-labelled endothelin-1 binding in cardiovascular tissues in a concentration-dependent manner (IC.sub.50 1.4 .mu.M in aortic smooth muscle, 0.8 .mu.M in ventricle membranes and 0.5 .mu.M in cultured aortic smooth muscle cells), but fails to inhibit binding to receptors in tissues in which ET.sub.B receptors predominate at concentrations up to 100 .mu.M. Cyclic pentapeptides related to BE-18257B, such as cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123), have been synthesized and shown to exhibit activity as ET.sub.A receptor antagonists (see, U.S. Pat. No. 5,114,918 to Ishikawa et al.; see, also, EP A10 436 189 to BANYU PHARMACEUTICAL CO., LTD (Oct. 7, 1991)). Studies that measure the inhibition by these cyclic peptides of endothelin-1 binding to endothelin-specific receptors indicate that these cyclic peptides bind preferentially to ET.sub.A receptors.
Development of non-peptidic ET antagonists has also become an important objective. Screening of several thousands of compounds from a chemical library for the ability to inhibit ET-1 binding to a human placental membrane preparation, identified a class of pyrimidinyl sulfonamides that weakly inhibit ET-1 binding. Modifications of these compounds led to the identification of a pyrimidinyl sulfonamide that inhibits ET-1 binding to ET.sub.A receptors at IC.sub.50 concentrations of about 0.2 .mu.M and to ET.sub.B receptors at concentrations of about 1 .mu.M (see, e.g., Canadian Patent Application 2,067,288; Canadian Patent Application 2,071,193; U.S. Pat. No. 5,208,243; and Clozel et al. (1993) Nature 365:759-761). The pyrimidinyl sulfonamide also exhibits in vivo activity in recognized animal models of vasoconstriction and has been deemed promising for the therapeutic treatment of vasoconstriction (Clozel et al. Clozel et al. (1993) Nature 365:759-761).
Screening of other compounds led to the identification of sulfathiazole as an inhibitor of the binding of endothelin to ET.sub.A (IC.sub.50 =69 .mu.m; see, Stein et al. (1994) J. Med. Chem. 37:329-331) and sulfisoxazole (IC.sub.50 &lt;1 .mu.M; see, Stein et al. (1994) J. Med. Chem. 37:329-331 but also see, co-owned U.S. application Ser. No. 08/065,202, which is herein incorporated by reference and to which this application claims priority). Particular N-(3,4-dimethyl-5-isoxazolyl)naphthalene-1-sulfonamides were shown to have endothelin antagonist activity. One derivative 5-diemthylamino-N-(3,4-dimethyl-5-isoxazolyl)naphthalene-1-sulfonamide is reported to have an IC.sub.50 value of 150 nM for inhibiting endothelin binding to ET.sub.A receptors and appears to exhibit oral activity in a rat model (see, Stein et al., 1994) J. Med. Chem. 37:329-331).
Endothelin antagonists and agonists as therapeutic agents
In view of the numerous physiological effects of endothelin and its apparent association with certain diseases, endothelin is believed to play a critical role in pathophysiological conditions, including hypertension, atherosclerosis, other vascular disorders, gastrointestinal disorders, renal failure, asthma, pulmonary hypertension, ischemia, coronary vasospasm, cerebral vasospasm and others (see, e.g., Saito et al. (1990) Hypertension 15: 734-738; Tomita et al. (1989) N. Engl. J. Med. 321:1127; Doherty (1992) J. Med. Chem. 35:1493-1508; Morel et al. (1989) Eur. J. Pharmacol. 167: 427-428). Because endothelin is associated with these and other disease states, more detailed knowledge of the function and structure of the endothelin peptide family should provide insight in the progression and treatment of such conditions.
To aid in gaining this understanding and to exploit the potential of endothelin as a therapeutic target, there is a need to identify additional compounds that modulate or alter endothelin activity. Compounds that modulate endothelin activity, particularly compounds that act as specific antagonists or agonists, may not only aid in elucidating the function of endothelin, but may be therapeutically useful. In particular, compounds that specifically interfere with the interaction of endothelin peptides with the ET.sub.A, ET.sub.B or other receptors should may aid in the design of therapeutic agents, and may be useful as disease specific therapeutic agents.
Therefore, it is an object herein to provide compounds that have the ability to modulate the biological activity of one or more of the endothelin isopeptides, It is another object to provide compounds that have use as specific endothelin antagonists. It is also an object to use compounds that specifically interact with or inhibit the interaction of endothelin peptides with ET.sub.A or ET.sub.B receptors as therapeutic agents for the treatment of endothelin-mediated diseases and disorders.